1. Harvest cells from a culture that is at or near peak density by centrifugation at 600 x g for 5 min. Pool the cell pellets into a single tube.
2. Adjust the concentration of cells to 2.0 x 10⁶/ml.xa0 If the concentration is too low, centrifuge at 600 x g for 5 minutes and resuspend the cell pellet with a volume of supernatant to yield the desired concentration.
3. Prepare a 15% (v/v) sterile DMSO solution in ATCC medium 1034 as follows:xa0 Add the required volume of DMSO to a glass screw-capped test tube and place on ice.xa0 Allow the DMSO to solidify.xa0 Add the required volume of refrigerated ATCC medium 1034.xa0 Dissolve the DMSO by inverting several times.xa0 If the DMSO solution is not prepared on ice, an exothermic reaction will occur that may precipitate certain components of the medium.
4. Mix the cell preparation and the DMSO in equal portions. Thus, the final concentration will be 10⁶ and 7.5% (v/v) DMSO. The time from the mixing of the cell preparation and DMSO stock solution before the freezing process is begun should be no less than 15 min and no longer than 60 min.
5. Dispense in 0.5 ml aliquots into 1.0 - 2.0 ml sterile plastic screw-capped cryules (special plastic vials for cryopreservation).
6. Place vials in a controlled rate freezing unit. From room temperature cool at -1°C/min to -40°C. If freezing unit can compensate for the heat of fusion, maintain rate at -1 C/min through heat of fusion. At -40°C plunge ampules into liquid nitrogen.
7. The frozen preparations are stored in either the vapor or liquid phase of a nitrogen refrigerator.
8. To establish a culture from the frozen state place an ampule in a water bath set at 35°C. Immerse the vial enough to cover only the frozen material. Do not agitate the vial.
9. Immediately after thawing, do not leave in the water bath, aseptically remove the contents of the ampule and inoculate into 5.0 ml of fresh ATCC medium 1034.
10. Incubate the tube on a 15° horizontal at 25°C with the cap screwed on tightly.

Schuster FL. Intranuclear virus-like bodies in the amoeboflagellate Naegleria gruberi. J. Protozool. 16: 724-727, 1969. PubMed: 5362388

Schuster FL, Clemente JS. 5-Bromodeoxyuridine-induced formation of virus-like particles in Naegleria gruberi EGs. J. Cell Sci. 26: 359-371, 1977. PubMed: 925099

Schuster FL, Dunnebacke TH. . Eur. J. Cell Biol. 14: 131-147, 1976.

Schuster FL, Dunnebacke TH. Growth at 37øC of the EGs strain of the amoeboflagellate Naegleria gruberi containing virus-like particles. II. Cytoplasmic changes. J. Invertebr. Pathol. 23: 182-189, 1974. PubMed: 4825255

Schuster FL, Dunnebacke TH. Virus-like particles and an unassociated infectious agent in amoebae of the genus Naegleria. Ann. Soc. Belge Med. Trop. 54: 359-370, 1974.

Dunnebacke TH, Schuster FL. An infectious agent associated with amebas of the genus Naegleria. J. Protozool. 21: 327-329, 1974. PubMed: 4838479

Dunnebacke TH, Schuster FL. The nature of a cytopathogenic material present in amebae of the genus Naegleria. Am. J. Trop. Med. Hyg. 26: 412-421, 1977. PubMed: 869097

De Jonckheere JF. A century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 41: 309-342, 2002.